The present invention relates generally to an automated cytopathology screening instrument and, more particularly, to a flow cytofluorometer or photometer for simultaneously obtaining multidimensional slit-scan type fluorescence or other photometric contours of particles, particularly biological cells, in flow.
A number of approaches have been developed directed to the problem of automating the process of analyzing cells from biological specimens. One particular, but not limiting, purpose is automated prescreening for gynecologic cancer and its precursors. A successful system must achieve a desired sensitivity to the few abnormal cells (have a low false negative rate), and at the same time maintain acceptable false positive rates. That is, the system must not generate an excessive number of false alarms which lead to the necessity of subsequent manual examination. These functional considerations should preferably be satisfied in a cellular screening system which has a high cell throughput, and which minimizes the amount of complex computation in the analysis required. This basically translates to a question of system resolution.
At one end of the resolution scale are high-resolution systems, for example utilizing a sub-micron scanning spot, wherein a full two-dimensional image of each cell is acquired and processed by a computer. This can involve relatively long data processing and computation times, and consequent low throughput. At the other end of the resolution scale are low-resolution systems wherein excitation and measuring apertures are larger than the cell of interest and a gross characteristics of each cell is examined, for example total fluorescence at a particular wavelength or light scatter at a particular angle. These systems permit measurements to be made at rates up to several thousand cells per second, and provide valuable information on specific cellular substances and parameters profiles on large populations of cells. However, they have thus far failed to demonstrate a capability to provide sufficient cellular information for the decisions required for application as a screening instrument.
Particular examples of high-resolution systems are disclosed in the Ehrlich et al U.S. Pat. No. 3,699,336 and in the Holm U.S. Pat. No. 3,918,812.
In low-resolution systems, a number of approaches have been proposed. The Coulter U.S. Pat. No. 2,656,508 describes the Coulter sensing principle which provides an indication of cell size. Of particular interest with respect to the present invention are the low-resolution systems of the following U.S. Patents, which systems generally optically view biological cells from two orientations or simultaneously examine a plurality of optical characteristics: Friedman et al U.S. Pat. Nos. 3,705,771, 3,785,735, and 3,788,744; and Fulwyler et al U.S. Pat. No. 3,710,933. As a particular example, the low-resolution system disclosed in the '933 Fulwyler et al patent includes a flow chamber through which cells in suspension flow sequentially in a stream, and a light beam which intersects the cell stream at right angles. The Fulwyler et al system measures small angle light scatter in one direction, and cellular fluorescence in another. An additional patent, Corll U.S. Pat. No. 3,910,702 describes a low-resolution system which is of interest with respect to certain particular aspects of the present invention in that an optical detector views particles along an axis of flow.
One particularly promising medium-resolution measurement technique suitable for use in the field of automated cytology is a slit-scan technique invented by L. L. Wheeless, Jr., and S. F. Patten, Jr. This technique is described in the Wheeless, Jr., et al U.S. Pat. No. 3,657,537 and in the literature reference: L. L. Wheeless, Jr., and S. F. Patten, Jr., "Slit-Scan Cytofluorometry", Acta Cytol., vol. 17, no. 4, pp. 333-339 (1973). These references provide the details of a static cell slit-scan cytofluorometer.
The slit-scan technique provides a more complete set of cellular parameters than is available with a low-resolution optical system, without producing the large data matrix associated with a high-resolution system. It represents a compromise solution to the problems of throughput and resolution.
In particular, the slit-scan configuration sequentially records the secondary fluorescence of an elongated portion of a cell (generally transversing the width of the cell) at discrete time intervals as that cell moves relative to the slit producing aperture. In a static cell instrument, this may be accomplished by recording the secondary fluorescence through a slit aperture at discrete intervals as that aperture is passed over the cell. This corresponds in a flow system to a recording of secondary fluorescence from a cell as it flows through a thin "wall" of excitation illumination. In both cases the slit aperture, or wall of excitation illumination, ideally is much smaller than the diameter of the cell of interest.
This type of medium-resolution slit-scan provides a graphic fluorescence contour, which in essence is a plot of the averaged fluorescence along the cell. From such contours, the fluorescence from the nuclei of the cells is readily distinguishable from cytoplasmic fluorescence.
The information from such a contour has been shown to be quite useful in prescreening. Specifically, for each cell, such characteristics as nuclear fluorescence, nuclear diameter, cytoplasmic diameter, cytoplasmic fluorescence, and nuclear to cytoplasmic diameter ratio (N/C ratio) may be determined. This technique is becoming increasingly useful since a comparison data base has been developed to enable the recognition of abnormal cells. In addition to the Wheeless and Patten "Slit-Scan Cytofluorometry" article identified above, the following literature references provide additional background information concerning the usefulness of the slit scan technique: L. L. Wheeless, Jr., and S. F. Patten, Jr., "Slit-Scan Cytofluorometry: Basis for an Automated Cytopathology Prescreening System", Acta Cytol., vol, 17, no. 5, pp. 391-394 (1973); L. L. Wheeless, Jr., S. F. Patten, Jr., and M. A. Cambier, "Slit-Scan Cytofluorometry: Data Base for Automated Cytopathology", Acta Cytol., vol. 19, no. 5, pp. 460-464 (1975); and M. A. Cambier, W. J. Christy, L. L. Wheeless, Jr. and I. N. Frank, "Slit-Scan Cytofluorometry Basis for Automated Prescreening of Urinary Tract Cytology" , J. Histochem. and Cytochem., vol. 24, no. 1, pp. 305-307 (1976).
In particular, from the Wheeless, Jr., Patten, Jr., and Cambier article entitled "Slit-Scan Cytofluorometry: Data Base for Automated Cytopathology", extensive investigation has shown that nuclear fluorescence from cells stained with Acridine Orange is higher in abnormal cells than in normal cells. The Cambier, Christy, Wheeless, Jr., and Frank article entitled "Slit-Scan Cytofluorometry Basis for Automated Prescreening of Urinary Tract Cytology", demonstrates potential usefulness in a slightly different area.
More recent slit-scan instruments utilize a flow cytofluorometer, details of which are described in: L. L. Wheeless, Jr., A Hardy and N. Balasubramanian, "Slit-Scan Flow System for Automated Cytopathology", Acta Cytol., vol. 19, no. 1, pp. 45-52 (1975). This flow cytofluorometer implements the flow system technique referred to in the Wheeless, Jr. and Patten, Jr. article "Slit-Scan Cytofluorometry", cited above, wherein secondary fluorescence is recorded from a cell as it flows through a thin "wall" of excitation illumination.
In this flow system, relatively transparent, fluorochrome stained cells in suspension ideally flow one-by-one through a focused slit of laser excitation light. Assuming the axis of flow is defined as the Z axis, in essence a planar sheet of excitation light in the X-Y plane is generated defining an excitation region. As the fluorochrome stained cells flow through the excitation region, fluorescence emissions are generated at the intersection of the planar excitation region and the cell. As the cell flows through the excitation region, a plurality of substantially planar parallel cross-sections of the cell along the Z axis are excited to secondary fluorescence. Monitoring the fluorescence emissions generates a slit-scan type contour along the Z axis.
It should be noted that one difference between the static slit-scan cytofluorometer previously described and the flow slit-scan cytofluorometer relates to the difference in the shape of the aperture effective cross-section. In the static cell slit-scan system, the aperture is a true slit which is passed across the fluorescence image of the cell. In the flow system, the sheet of laser light has a Gaussian intensity distribution, in the direction of flow (along the Z axis), and is elliptical in cross-section (in the X-Y plane). This difference is insubstantial in practice, and the expression "slit-scan type contour" as employed herein is intended to refer interchangeably to contours generated by either of these approaches.
One particular problem in evaluating cells by means of a one-dimensional slit-scan type contour along a single axis is that false alarms may occur as a result of such factors as cell orientation. It has been determined that the majority of false alarms are due to multinucleate cells or overlapping cells oriented such that both nuclei enter the measurement region simultaneously. Such a cell or cells may be completely normal but, in this orientation, exhibit greater nuclear fluorescence than uninuclear cells of the same cell type, and thus be erroneously classified as abnormal. Similarly, the entire cell may be oriented with the plane of the cell parallel to the plane of the slit excitation such that there is substantially no discrimination between cytoplasmic fluorescence and nuclear fluorescence. The problem of binucleate cells is discussed in particular in the literature reference: J. L. Cambier and L. L. Wheeless, Jr., "The Binucleate Cell: Implications for Automated Cytopathology", Acta Cytol., vol. 19, no. 3, pp. 281-285 (1975).
At least four general approaches to solving cell classification problems resulting from particular cellular orientations have been proposed. A first technique is the use of flow nozzles and analysis chambers which tend to produce a desired orientation of the cells. This technique is suggested by the Hogg U.S. Pat. No. Re. 29,141, and in the literature reference: D. Kay and L. L. Wheeless, Jr., "Experimental Findings on Gynecologic Cell Orientation and Dynamics for Three Flow Nozzle Geometries", J. Histochem. Cytochem., vol. 25, no. 7, pp. 870-874 (1977).
A second technique is to provide a second analysis stage which analyzes cells which have recorded a positive event as a result of passage through a first slit-scan type measurement system. The second stage may employ a higher resolution analysis, and may obtain and analyze a two dimensional image. Such techniques are generally described in the following two literature references: L. L. Wheeless, Jr., D. B. Kay, M. A. Cambier, L. Cambier and S. F. Pattern, Jr., "Imaging Systems for Correlation of False Alarms in Flow", J. Histochem. Cytochem. vol. 25, no. 7, pp. 864-869 (1977); and D. B. Kay, L. Cambier and L. L. Wheeless, Jr., "Imaging System for Correlating Fluorescence Cell Measurements In FLow", Proceedings of the Society of Photo-Optical Instrumentation Engineers, San Diego, Calif., vol. 126, Clever Optics, pp. 132-139 (Aug. 25-26, 1977). (It should be noted that a second stage may still be required with the present invention, but there will be fewer false alarms necessitating second state processing.)
Third, and particularly in the context of a second analysis stage as mentioned immediately above, it has been suggested that a segmented slit technique would be useful. (Wheeless, Kay, Cambier, Cambier, and Patten, "Imaging Systems for Correlation of False Alarms in Flow", above.) This technique produces a plurality of slit-scan type contours, each representing fluorescence across only a portion of the cell, and may be considered a low-resolution form of two-dimensional imagery.
Fourth, it has been recognized that apparatus which would provide one-dimensional slit-scan contours along three orthogonal axes would be quite useful in providing additional information useful in reducing the incidence of false positive indications, particularly those resulting from binucleate or overlapping cells which are oriented such that in a single slit-scan type contour each appears to be an abnormal cell having a high nuclear fluorescence, rather than a binucleate cell or a pair of overlapping cells producing two distinct peaks on the slit-scan contour. The Cambier and Wheeless, Jr. article entitled "The Binucleate Cell: Implications for Automatic Cytopathology", cited above, itself refers to the desirability of a system employing three orthogonal slits. An additional such reference is in the Wheeless, Jr., Hardy, and Balasubramanian article, also cited above, which describes a "Slit-Scan Flow System for Automated Cytopathology". While not prior art with respect to the present invention, a more recent analysis of false alarms may be found in L. L. Wheeless, Jr., J. L. Cambier, M. A. Cambier, D. B. Kay, L. L. Wightman and S. F. Patten, Jr., "False Alarms in a Slit-Scan Flow Systems: Causes and Occurrence Rates Implications and Potential Solutions" , J. Histochem. Cytochem., vol. 27, no. 1, pp. 596-599 (1979).
In particular multi-dimensional slit-scan apparatus such as has been proposed, three successive excitation laser beams are positioned sequentially along the flow axis, oriented in mutually orthogonal planes.
In a multi-dimensional slit-scan flow system, in which three orthogonal projections of cell fluorescence are collected, overlapping cells produce three slit-scan contours. In at least one of these contours the nuclei will in most cases be evidenced by two distinct fluorescence peaks, which can be recognized as such, and the nuclear fluorescence measurement disregarded.
Two particular problems arise with such a sequential multiple slit-scan type instrument. Perhaps the more serious is that cell orientation and/or overlap may change from one measurement station to the next, and the result that the contours are not truly orthogonal, and, depending upon the precise nature of the tumbling between stations, two of the slit-scan contours may actually be along the same axis of the cell. Cell orientation problems in the context of a system employing three orthogonal slit measuring beams are discussed in particular in the reference D. B. Kay and L. L. Wheeless, Jr., "Laser Stroboscopic Photography Technique For Cell Orientation Studies in Flow", J. Histochem. Cytochem., vol. 24, no. 1, pp. 265-268 (1976). Another problem is that a plurality of cell excitations are required, with the result that the cell may become bleached, thus distorting the measurements.